The introduction of biologically active molecules, such as for example, DNA, RNA or proteins, into living cells is an important instrument for studying biological functions of these molecules. A preferred method for introducing foreign molecules into the cells is electroporation which, unlike other methods, only causes slight permanent changes to the biological structure of the target cell by the transfer reagents. During electroporation the foreign molecules are introduced into the cells from an aqueous solution by a brief current flow wherein the cell membrane is made permeable for the foreign molecules by the action of short electrical pulses. As a result of the “pores” briefly formed in the cell membrane, the biologically active molecules initially enter the cytoplasm in which they can already exert their function to be studied if necessary. In cases where DNA is introduced into eukaryotic cells, this must enter the cell nucleus however, so that it is possible for the genetic information to be expressed. In the case of dividing cells, this can take place during the cell division wherein the DNA passively enters the cell nucleus after the temporary dissolution of the nuclear membrane. In studies of quiescent or weakly dividing cells, for example, primary animal cells, however, the DNA does not enter into the cell nucleus in this way so that corresponding methods cannot be used here or at least are very tedious. Moreover, especially when DNA is introduced into animal cells, so-called transfection, particular problems frequently occur as a result of the lability of the cells, since the survival rate of the cells influences the efficiency of the transfection as an important parameter.
In the past cell culture media (Anderson et al. (1991), J. Biochem. Biophys. Meth. 22, 207) or salt solutions buffered with phosphate or HEPES (Potter et al. (1984), Proc. Natl. Acad. Sci. USA 81, 7161; Fromm et al. (1985), Nature 319, 791) were frequently used to take up the animal cells and DNA molecules. However, non-buffered or weakly buffered mannitol or saccharose solutions were also used during the electroporation of animal cells (Shimizu et al. (1986), Med. Immunol. 11, 105; Riggs et al. (1986), Proc. Natl. Acad. Sci. USA 83, 5602). Such non-buffered or weakly buffered solutions leads to an increased cell mortality and significantly reduced transfection efficiency as described in Yan et al. (1998), BioTechniques 24, 590.
Yan et al. (1998) describes the use of a buffer solution for electroporation consisting of 100 mM HEPES, 137 mM NaCl, 4 mM Na2HPO4 and 6 mM dextrose. Smooth muscle cells from human aorta could certainly be successfully transfected in this buffer but the transfection efficiency was only 15% with a survival rate of only 10 to 20%. Furthermore, the buffer used by Yan et al., is only optimised for voltage pulses up to 500 V so that no indication is given as to whether this buffer can also be used for higher voltage pulses such as are required for the direct transfection into the cell nucleus. In any case, however, the transfection efficiencies achieved in this buffer are too low to meet higher demands.
In many cases, buffers having a low ionic strength and therefore low conductivity were used in order to avoid cell damage as a result of high currents which was observed when using buffer solutions having high conductivity, especially during the application of longer high-voltage pulses.
Friedrich et al., 1998 (Bioelectrochem. and Bioenerg. 47, 103) describes that the current flow during electroporation leads to a change in the pH in the vicinity of the electrodes as a result of electrolysis of the water. This change in the pH causes the release of cytotoxic Al3+ ions from the aluminium electrodes of the cuvettes and therefore causes increased cell mortality. The authors here propose a shortening of the pulse duration to increase the transfection efficiency. No information is given on any change to the buffer used.
Divalent cations such as Mg2+ for example, can be added to the electroporation buffer. Magnesium ions facilitate the binding of DNA to the surface of the cells and thereby bring about an increased transfection rate. However, this only seems to apply for Mg2+ concentrations up to 10 mM since at higher concentrations negative effects predominate, such as, for example, a reduction in the electrophoresis as a result of neutralisation of the charges of the DNA molecules or heating of the buffer as a result of an increase in the conductivity as described in Xie and Tsong (1993), Biophys. J. 65, 1684 and Neumann et al. (1982), EMBO J. 1, 841; Klenchin et al. (1991), Biophys. J.60, 804.
Furthermore, the buffer can be matched with regard to its composition to the intracellular conditions in order to increase the survival rate of the cells. Thus, a buffer having high potassium and low sodium concentrations corresponding to the cytoplasm can be used so as to prevent any collapse of the intracellular Na+/K+ ratio as a result of substance exchange via the pores formed in the cell membrane during the electroporation as described in van den Hoff et al. (1995), Methods in Mol. Biol. 48, Chapter 15, 185-197. However, this results in a reduction in the transfection efficiency for pulses having a field strength higher than 1300 V/cm.
All the buffer solutions described so far however have the disadvantage that the transfection efficiencies achieved when using them are relatively low and/or the buffers are not suitable for application during the electroporation of quiescent or weakly dividing cells.